Project Summary Glutamate transporters pump the neurotransmitter from the synaptic cleft into the cytoplasm of glial cells and neurons against concentration gradients reaching a million-fold by harnessing the energy stored in the form of the trans-membrane electrochemical gradients of ions. Specifically, they couple uptake of each molecule of glutamate to the symport of three sodium ions and a proton and to the antiport of a potassium ion. Their dysfunction is associated with a range of neurological disorders and the extensive brain damage following traumatic injury and stroke. The complete transport cycle of glutamate transporters involves binding of the substrate and the symported ions to the outward facing conformation of the transporter, isomerization of the transporter into the inward facing conformation, the release of the substrate and ions into the cytoplasm, binding of the counter-transported potassium ion and the return of the transporter into the outward facing state. The structural information on this family comes from the crystallographic studies on a bacterial homologue, GltPh, which couples aspartate uptake to the symport of three sodium ions, but not to the movements of other ions. During our previous funded period, we have primarily focused on the large-scale conformational transitions of GltPh that underlie the trans-membrane translocation of the substrate and coupled ions. We now seek to investigate at the structural and mechanistic level the events associated with the substrate and ions binding and release on the two sides of the membrane that are at the heart of the functional specificity of the transporters. We further propose to employ mutagenesis to reconstitute in GltPh coupling to protons and potassium ions observed in the mammalian transporters. Finally, we propose to effectuate in GltPh a switch of specificity from employing sodium gradients to using protons to drive transport by mimicking the amino acid composition of the ion-binding sites of the proton-coupled members of the family. By combining these studies with the structural studies on the bone fide proton coupled bacterial glutamate transporters, we aim to understand how the conserved protein architecture and the structural mechanisms are adapted to allow functional diversification. Our long-term goal is to achieve a complete mechanistic description of the catalytic cycle of these transporters and to gain rational control over their functional properties.